amplicon sequencing with Lib-L (Junior)

[audioPL]

Hi all,

I would like to run sequencing on a multiplexed probe that contain 100-1000 amplicons, generated with the use of a single primer (amplicon structure is: P===P). One option is to ligate RL adaptors and proceed with Lib-L procedure. I am afraid, however, that during RL preparation, short amplicons will be lost. Thus, I think that the better way will be to add Lib-L primers (Lib-LA/Lib-LB) to my amplicons directly during PCR. The problem is that I can’t differentiate primers P in the reaction, but the amplicons for Lib-L sequencing should have a AP===PB structure. Could someone confirm that a combination of AP/BP primers in the PCR will solve the problem? How exactly RL kit works? Is the combination of Lib-LA/Lib-LB adaptors used in the kit and are they randomly ligated to blunt-ended fragments? If so, this would be exactly what I can expect in my PCR with AP/BP primers and only products with AP===PB structure (Lib-LA===Lib-LB from RL) will be amplified in emPCR. Can someone confirm this?

Bests,
Mirek

[ajthomas]

If I understand correctly, you have the same primer on both ends, right? If that's the case, then integrating the adapters into the primers won't work well. You could still do it, but your titer would be lower and perhaps somewhat variable. Only about half of your PCR products would be functional in emPCR. That might be just fine and your sequencing would work just fine, but you won't know for sure until you try.

The RL kit works by ligating on Y-shaped adapters onto each end. The open end of the Y has the A adapter sequence on one side and the B adapter sequence on the other. Once ligated on, each strand has A on one end and B on the other. That should work just fine in your situation. You say you're concerned about losing the short amplicons. How short are they? If they are too short, you will lose them in the size selection step of the RL protocol if you follow the standard procedure. You could modify that step to lower the size selection threshold if that's a problem.

I think if it were me, I would try putting the adapters on the primers first because that's much simpler and more cost effective. Give it a try and see if you get acceptable results. If not, then try the RL kit.

Back to the short amplicon issue...if your short amplicons are too short, and/or if the size difference between the short and long amplicons is too great, your emPCR might give you problems. You may get goo much amplification of the short fragments and not enough of the longer ones and you will get mostly short fragment sequences in the final results.

[audioPL]

Dear ajthomas, thank you for your reply! You are right, I have the same primer on both ends. I understand that by chance only half of PCR products will be used in emPCR, I think I will try it.

Regarding the size of the amplicons - the shortest are about 150pb, longest about 600-700bp but the size distribution vary from sample to sample. I know thai it can be problematic. The other option (than direct sequencing of amplicons) I consider is a random concatenation of all amplicons and then nebulization+RL+Lib-L. This is more indirect, more tricky and require more bioinformatics at the end to split reads from concaterized fragments (I am working on non-model species) so I tried not to go that way. What do you think, do you see another option in my case?

Thank you for clarification regarding the RL adapters; unfortunately I couldn't find it in any brochures. Do you have more information/experience how to lower the size selection threshold during RL preparation? Dilute the sizing solution? With, how? ;-)

Bests,
Mirek

[ajthomas]

No, I don't see another option in your case, at least not one worth mentioning. I think your best options are to sequence the amplicons directly or ligate adapters on. As for the size range issue, it won't make any difference which option you choose; either one will yield about the same size distribution.

It turns out that your size distribution is about the same as what I'm doing. I have amplicons ranging from about 150-400, with another that I sequence on occasion that is about 650. These sizes are without the adapter and primer sequence. Once those are added, the shortest one is about 270 or so. If yours are 150 with the primers included, they might be cause a problem, but all that would happen is that you would get more of those than the others, and the signals would probably be somewhat stronger. It's probably best to just try it and see.

After amplifying my amplicons, I purify them with AMPure beads. My amplification reactions are 25 ul, and I have found that if I use 18-20 ul of beads, I get rid of any primer dimers and leave behind my amplicons. You can adjust the size cutoff by changing the volume of AMPure beads you use (ratio of volumes of DNA/beads). More beads lowers the cutoff and vice versa. I figured out the right amount to use by just using different amounts of beads with a ladder and then finding the amount with the right cutoff. Different lots of beads will have slightly different performance, so you will want to figure out the right amount for yourself.

[pmiguel]

Thank you for clarification regarding the RL adapters; unfortunately I couldn't find it in any brochures.

It's because Roche decided not to share the nature of their RL adapters with their customers. Basically they let slip that they were Y-adapters (as if that wasn't obvious anyway.) But the more bizarre aspect turned out to be that they created the complementary (stem) region by placing inosines at positions that did not match.

Seems like it would have been easier to just use the MID's as stems and choose only palindromic ones.

--
Phillip

[audioPL]

ajthomas thanks for your help! this week I'll have results. bests, mirek

[audioPL]

It's because Roche decided not to share the nature of their RL adapters with their customers. Basically they let slip that they were Y-adapters (as if that wasn't obvious anyway.) But the more bizarre aspect turned out to be that they created the complementary (stem) region by placing inosines at positions that did not match.

Seems like it would have been easier to just use the MID's as stems and choose only palindromic ones.

--
Phillip

Thx. Do you know where exactly the inosines are located, where/how long is the stem? Do you know the sequences of RL adapters used in the kit? Is the sequence of one arm of Y-adapter 5'-3' PrimerA and the second 3'-5' complementary to PrimerB? Probably this is the only option possible, right?

bests,
mirek

[zhengz]

Hi audioPL,

There is a protocol "Titration-free 454 sequencing using Y adapters". http://www.nature.com/nprot/journal/....2011.369.html

Not exactly as the Roche adapter, but might be of your interest?

[audioPL]

Hi audioPL,

There is a protocol "Titration-free 454 sequencing using Y adapters". http://www.nature.com/nprot/journal/....2011.369.html

Not exactly as the Roche adapter, but might be of your interest?

Hi zhengz,

Looks interesting, thanks! m

[ECO]

Thx. Do you know where exactly the inosines are located, where/how long is the stem? Do you know the sequences of RL adapters used in the kit? Is the sequence of one arm of Y-adapter 5'-3' PrimerA and the second 3'-5' complementary to PrimerB? Probably this is the only option possible, right?

bests,
mirek

Go here: http://www.idtdna.com/catalog/fusion...s/default.aspx

...and read up...I believe the link on the lower right is what you want. If you add a mid to cart, you'll be able to see the sequences of both oligos and where the MID and iosines are located.